H2S catalysed by CBS regulates testosterone synthesis through affecting the sulfhydrylation of PDE

Abstract Testosterone deficiency resulted in increased mortality in men. Our previous work found that hydrogen sulphide (H2S) significantly alleviated the spermatogenesis disorder. To investigate whether H2S could regulate testosterone synthesis and the relative signalling pathways. Disorder model of testosterone synthesis was constructed in vitro and in vivo. The cell viability was detected using CCK‐8 method. The concentration of H2S and testosterone were examined using ELISA kits. The relative mRNA and protein expression of CBS, PDE4A, PDE8A and proteins related to testosterone synthesis were detected by RT‐qPCR and western blotting. PAS staining was used to detect the inflammatory status of testis. The sulfhydryl level of PDE4A and PDE8A was determined by Biotin Switch Technique. CBS overexpression inhibited while knockdown promoted LPS + H2O2 induced injury in testosterone synthesis of MLTC‐1 cells, though regulating the level of H2S. The LPS + H2O2 induced inhibition on cAMP and p‐PKA was recovered by CBS overexpression, while addition of the specific inhibitor of PKA had opposite effects. CBS overexpression alleviated the inflammation status in testis and promoted the expression of StAR, P450scc, P450c17 and 3β‐HSD. CBS could also exhibit its protective role through promoting sulfhydrylation of PDE4A and PDE8A. H2S catalysed by CBS could recover testosterone synthesis in vitro and in vivo through inhibiting PDE expression via sulfhydryl modification and activating cAMP/PKA pathway.


| INTRODUC TI ON
Testosterone is the most important reproductive hormone in men, which is mainly synthesized by male testis and plays an important role in the growth, development, reproduction and maintenance of organ function. Testosterone deficiency can seriously affect male reproductive and sexual functions and cause abnormal glucose metabolism and dysfunction of cardiovascular system, resulting in decreased quality of life and increased mortality. [1][2][3] Studies have shown that the incidence of hypogonadism and low testosterone levels is about 2.1%-5.7% in men aged 40-79 years. 4 For a long time, androgen replacement therapy has been the main means to solve the insufficient synthesis and secretion of testosterone, which can alleviate the dysfunction caused by testosterone deficiency. However, androgen replacement therapy has strict contraindications and needs to be monitored for a long time in the course of treatment, while long-term use of androgen brings the patients with possible cancer and cardiovascular risks, as well as other adverse reactions. 5,6 Therefore, it is of great significance to investigate new mechanisms involved in the regulation of testosterone synthesis and find potential new targets for endogenous testosterone regulation.
Hydrogen sulphide (H 2 S), an important gas signalling molecule involved in the regulation of physiological functions, has become one of the research hotspots in recent years. H 2 S in human body mainly catalyses l-cysteine production by Cystathionine γ-lyase (CSE) and Cystathionineβ-synthase (CBS) and plays a key role in a variety of signal pathways that regulated physiological functions. 7 In the male reproductive system, CBS is mainly found in the Leydig cells of the testis, and CSE is mainly distributed in Sertoli cells and immature spermatocytes including spermatogonia. 8 Our previous work found that H 2 S could significantly alleviate the spermatogenesis disorder caused by inflammation and oxidative stress, among which H 2 S derived from CBS might be one of the key factors to maintain testicular function. 9 H 2 S can play a physiological role by regulating protein post-translational sulfhydration. H 2 S targets the sulfhydryl group (Cys-SH) on the side chain of cysteine residues to achieve sulfhydryl (-SH)-disulphide bond (-S-S-) conversion, which plays a key regulatory role in a variety of physiological and pathological processes. 10,11 Phosphodiesterase (PDE) is one of the key targets in regulating the cAMP/PKA pathway of testosterone synthesis. Inhibiting the activity of PDE is of great significance in solving testosterone secretion disorders. Several studies have shown that H 2 S was a non-specific inhibitor of PDE and participated in the regulation of cAMP-dependent signalling pathways. 7 Since PDE proteins contain a large number of cysteine residues, it is speculated that the mechanism by which H 2 S inhibits PDE activity may be related to sulfhydryl sulphide modification. 7 However, the significance of H 2 S for male reproduction still remains unclear.
In this study, we intended to clarify the new mechanism of H 2 S regulating testosterone synthesis through the modification of PDE sulfhydryl sulphide using mouse Leydig Tumour Cells (MLTC-1) and inflammation-induced testosterone synthesis disorder mouse model, aiming at providing theoretical basis for the treatment of testosterone deficiency. Subsequently, all the groups except control group were added with LPS (10 μg/mL) and H 2 O 2 (250 μmol/L) for 12 hours to construct disorder model of testosterone synthesis.

| Recombinant vector constructs
Mouse CBS cDNA, PDE4A cDNA and PDE8A cDNA clone were designed via GenBank database and synthesized by Nanjing Jiancheng company (Nanjing, Jiangsu, China) and then was sequenced for veri-  and then treated with LPS and GYY4137 as per the above procedure.

| Cell viability assay
Cell viability was measured using a CCK-8 kit (KeyGen Biotech, Nanjing, China). The cells were plated into 24-well plates at a density of 1 × 10 5 cell per well and incubated for 24 hours, followed by addition of CCK-8 solution. The viable cells were quantified at 450 nm by a Microplate Reader (Potenov, Beijing, China).

| ELISA assay
The level of H 2 S for mice, testosterone and the activity of PDE was determined via rat-specific enzyme-linked immunosorbent assay (ELISA) commercial kits from My BioSource (San Diego, CA, USA).

| Western blotting
Western blotting was performed for the detection of protein levels based on as per the reported method with little modification. 12 The tissue samples or cells were digested by RIPA solution (Solarbio, Antiβ-actin (ab8226, 1:500) that selected as a control, followed by secondary antibody Goat Anti-Mouse IgG H&L (HRP) (ab6728, 1:2000). All the antibodies used were purchased from Abcam Co., Ltd. (Cambridge, USA). The density of protein bands was examined using an Image analyser quantitative system.

| Biotin Switch Technique (BST)
The collected cell samples were lysed with HEN buffer (250 mmol/L Hepes-NaOH, pH 7.7, 1 mmol/L EDTA, 0.1 mmol/L neocuproine, 100 µmol/L deferoxamine) and protease inhibitors. The proteins in cell lysates were detected using a BCA kit and were incubated with Biotin-HPDP (10 mmol/L, Invitrogen, CA, USA) for 3 hours, followed by 20 μL of streptavidin-agarose beads (Invitrogen, CA, USA) for 18 hours on a roller system at 4°C. The beads were washed twice with ice-cold HEN buffer solution. The level of sulfhydryl groups in PDE protein was analysed by Western blotting mentioned above and probed with mouse antibody against PDE4A (ab200383, 1:1000) and PDE8A (ab109597, 1:1000).

| Real-time reverse transcription quantitative PCR (RT-qPCR)
The RNAiso Plus Kit (Takara, Dalian, China) was used to extract the total RNA in tissues, and Reverse Transcription kit (Thermo Scientific, Waltham, USA) was employed to transcribe the RNA into cDNA, after which cDNA product was stored at −20°C. The measurement of mRNA expression level was performed on a real-time fluorescent quantitative PCR system (Roche Light Cycler96, Roche, Indiana, USA).
The RT-qPCR amplification of cDNA samples was performed under the following conditions: pre-denaturation at 95°C for 3 minutes (1 cycle); amplification at 95°C for 5 seconds, 60°C for 30 seconds and 72°C for 45 seconds (40 cycles); the last cycle was processed at 72°C for 10 minutes. The expression of the target genes was normalized to the level of internal NAPDH using the 2 −ΔΔCt method.

| Periodic acid-Schiff staining (PAS)
The testicles samples were cut into 3 μm slices and then fixed in 10% buffered neutral formalin for histopathological staining. Tissue samples were dehydrated and embedded in paraffin. The slices were mounted on glass layers and stained with Hematoxylin and Eosin (HE). 13

| Statistical analysis
All the data were performed in the form of mean ± standard deviation with each experiment repeated at least three times. Student's t-test was used for comparison between the two groups of data, and Tukey's test for one-way ANOVA was used for comparison between multiple groups using Prism software version 8.0. The P-value less than .05 was regarded as significantly different.

| H 2 S pretreatment reduced LPS + H 2 O 2 induced injury in testosterone synthesis of MLTC-1 cells
Cell viability result presented in Figure 1A

| CBS overexpression inhibited LPS + H 2 O 2 induced injury in testosterone synthesis through promoting sulfhydrylation of PDE4A and PDE8A
PDE4A and PDE8A overexpression models were constructed to verify their relationship between CBS ( Figure 3A). As shown in Figure 3B

| CBS overexpression in vivo inhibited LPS + H 2 O 2 -induced injury in testosterone synthesis through promoting sulfhydrylation of PDE4A and PDE8A
PDE4A and PDE8A overexpression in vivo models were constructed to further verify their relationship between CBS ( Figure 5A). As shown in Figure 5B

| D ISCUSS I ON
Testosterone deficiency exhibits negative impact on male reproductive and sexual functions, and one of the common causes of testosterone deficiency was testosterone synthesis affected by gene, ageing or infections of male reproductive system, which are universally accompanied by inflammatory and oxidative damage. [14][15][16] Improving the synthesis of endogenous testosterone may be of important implications for reducing the potential risks associated with androgen replacement therapy. 17,18 The classical molecular signalling pathway for testosterone synthesis is luteinizing hormone/luteinizing hormone receptor/cyclic adenosine monophosphate/protein kinase A (LH/LHR/cAMP/PKA).
Activation of the PKA signalling pathway causes the initiation of a variety of transcription factors that regulate testosterone synthesis is that H 2 S can inhibit the activity of PDE2A and activate cAMP/ PKA-dependent cellular energy metabolism. 28 However, the significance of H 2 S in male reproduction has not been clear.
Our previous work found that H 2 S could significantly reduce the spermatogenesis disorder caused by inflammation and oxidative stress, among which H 2 S from CBS might be one of the key factors to maintain testicular function. 9 In this study, we further investigated the effect of H 2 S caused by CBS overexpression or inhibition on Finally, we investigated the mechanism by which H 2 S downregulated the expression of PDE4A and PDE8A. Using BST method, the inhibitory effects that H 2 S exhibited resulted from its modification of PDE, since sulfhydryl level of PDE4A and PDE8A was increased with CBS overexpression or H 2 S reaction time promoted. Other recent studies have also confirmed that H 2 S could modify PDE through sulfhydryl modification, and then increase cAMP level. 30 Hence, H 2 S played a physiological role on downregulating PDE level via

ACK N OWLED G EM ENTS
We would like to acknowledge everyone for their helpful contributions on this paper.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests. The levels of relative mRNAs related to testosterone synthesis were detected by RT-qPCR. *P < .05, *P < .01 compared with control group, #P < .05, #P < .01 compared with model group, %P < .05, %%P < .01 compared with model + OE-CBS group, NS represented no significance visualization (equal). Xia Zhao: Formal analysis (equal); investigation (equal); methodology (equal); software (equal); supervision (equal).

E TH I C A L A PPROVA L
The research protocol was reviewed and approved by the Ethical Committee and Institutional Review Board of the Zhongda Hospital, School of Medicine, Southeast University.

CO N S E NT TO PU B LI S H
All of the authors have consented to publish this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are free access to available upon request.